Immunosorbents are useful in biochemical processes and applications, such as the isolation and purification of proteins, immunodiagnostics, and as sensors for detecting the presence of biochemical substances. Immunosorbents are capable of isolating and purifying both proteins and haptens present in complex mixtures at extremely low concentrations. [Generally, proteins are polymers formed from at least one hundred amino acid residues joined by amide linkages, and peptides are comprised of from two to ten amino acid residue joined through amide linkages; for the sake of simplicity, the term protein is used hereinafter to describe any molecule containing two or more amino acid residues joined through amide linkages. Haptens are non-proteinaceous molecules which have a sufficiently recognizable chemical structure that an antibody will have an avidity for the compound.]
Immunosorbents are generally made by immobilizing an antibody on a suitable support (also referred to as a substrate or matrix), such as a gel, membrane or other suitable chromatographic support material. Since antibodies have both high specificity and binding affinity for particular antigen or hapten molecules, immunosorbents are frequently used to separate and purify specific antigen or hapten molecules from dilute solutions thereof by interaction with immobilized antibodies on the immunosorbent matrix. Immunosorbents are also used in immunodiagnostics to assay quantities and types of proteins present in biological and biochemical samples. Of course, other uses of immunosorbents are known or will be apparent to those of skill in the art.
The total binding capacity of an immunosorbent is determined by the number of active antigen binding sites; unfortunately, only a small fraction of the antibodies immobilized on the matrix of currently available immunosorbents are "active" (i.e., able to bind to antigens). It is believed that interactions between the antigen binding site (antitope or F.sub.ab) on the antibody and the support interfere with the ability of the bound antibody to attract and bind antigens which can be bound when the antibodies are not bound.
Typical immunosorbents, having antibodies bound directly to a matrix, bind less than 30% of the theoretical capacity of antigen or hapten molecules based on a 1:2 stoichiometry of antibody to antigen. [In other words, less than 30% of the bound or immobilized antibodies are oriented so as to have an avidity for antigens having the same epitope as the antigens eluted therefrom]. This results in a great waste of bound antibody, and requires the use of much larger quantities of immunosorbent material to achieve good separation and purification of desired compounds.
According to Cuatrecasas, the use immunosorbents, (immunoadsorbents) for the purification of antibodies or antigens is related to affinity chromatography. See "Protein Purification By Affinity Chromatography," J. Bio. Chem, Vol. 245, No. 12, Jun. 25, 1970, pp. 3059-3065. Both technologies involve the separation of specific molecules or bioaffinants from solutions through attraction to and binding by specific molecular recognition sites on stationary bioaffinity ligands attached to a support. In the case of immunosorbents, generally, the bound ligand is an antibody, and the specific molecular recognition sight on the antibody is referred to as an antitope; antigens for which the antitope of the antibody has an avidity for are said to contain the epitope for the antibody antitope. In certain instances, it is possible to synthesize the epitope for a given antitope of an antibody. This is especially useful when the antigen is expensive, in short supply, or difficult to work with.
Cuatrecasas has attributed the limited success of immunosorbents in purifying antibodies and antigens on the lack of suitable solid supports for the attachment of ligands, and on the failure to fix these ligands at a sufficient distance from the matrix backbone. Therefore, it was proposed that bioaffinity matrices and immunosorbents be prepared with the ligand groups (antibodies), critical in the interaction with the macromolecule to be purified (antigen), sufficiently distant from the backbone of the solid matrix to minimize allosteric (steric) interference with affinity processes. Hence, a "spacer arm" was used to attach ligands to a solid support; the spacer arm maintains the bound or immobilized ligand at a sufficient distance from the solid matrix so as to minimize matrix effects on the binding ability of the bound ligand. However, the use of such spacer arms increases the cost and difficulty of producing bioaffinity matrices and immunosorbents.
Immobilization of an antibody on an immunosorbent matrix is usually achieved by interaction between the antibody and the matrix. Frequently, a matrix is activated so that the matrix readily binds to reactive amine functional groups in the protein chain forming the antibodies. Thus, the antibodies are bound to the matrix and immobilized.
For example, Goding discloses that reactive groups such as cyanogen bromide (CNBr), N-hydroxysuccinimide, carbonyldiimidazole, and toluene sulphonylchloride can be used to activate a matrix, and the activated matrix can then be used to immobilize antibodies through accessible primary amino groups on the protein chain forming the antibody. See Monoclonal Antibodies: Principles And Practice, Academic Press, New York (1983). Because amino groups can be present on various parts of the protein chain (or chains) forming the antibodies, the orientation of proteins immobilized in this way on activated matrices is thought to be random on the immunosorbent support matrix surface. Thus, a significant portion of the distribution of orientations of antibodies on the immunosorbent surface obtained by random amino-coupling chemistry can be expected to lower avidity and/or completely obstruct the antigen-combining sites. Thus, attempts to follow the suggestions of Cautrecasas, discussed above, by adding a "spacer arm" between an immunosorbent support matrix and a bound antibody have not been successful in increasing the activity of immunosorbents. It is believed that a significant portion of the bonds between the "spacer arms" and the bound antibodies interfere with the ability of the bound antibodies to attract and bind antigens.
For example, O'Shannessy et al, in "Specific Conjugation Reactions Of The Oligosaccharide Moieties Of Immunoglobulins," J. Appl. Biochem., 7, 347-355 (1985), discloses that immunoaffinity supports can also be prepared by coupling the carbohydrate moieties of certain antibodies to a matrix. For example, a polyclonal IgG (immunoglobulin) fraction of goat anti-human albumin was coupled to AAH (agarose adipic acid hydrazide). However, this method may not be generally useful for preparing immunosorbents, since certain antibodies, such as IgG, have low carbohydrate content. Further, it is believed that immunosorbents prepared by this method will suffer the same problem as immunosorbents prepared by other techniques, such as the loss of immunologic activity due to binding of antibody through or in close proximity to the antigen binding sites and the multiple orientations of the antibody molecules on the matrix.
It is also possible to immobilize antibodies on an immunosorbent support matrix through non-covalent bonding; for example, an immunosorbent support matrix of polystyrene will form noncovalent bonds between the long alkyl chains of isoleucine in proteins. The long non-polar alkyl chains readily associate with the non-polar environment of the plastic matrix and are attached to the polystyrene matrix due to low energy forces.
For further examples of the synthesis, variety and effectiveness of immunosorbents, their applications and for additional background, see: Comoglio, S., Massaglia, A., Rolleri, E. and Rosa, A. (1976) "Factors affecting the properties of insolubilized antibodies." Biochimica et Biophysica Acta, 420, 246-257. Cress, M. C. and Ngo, T. T. (1989) "Site Specific Immobilization of Immunoglobulins." Amer. Biotech. Lab., xx, 16-19. Eveleigh, J. W. and Levy, D. E. (1977) "Immunochemical characteristics and preparative application of agarose-based immunosorbents." J. Solid-Phase Biochem., 2(1), 45-77. Gersten, D. M. and Marchalonis, J. J. (1978) "A rapid, novel method for the solid-phase derivatization of IgG antibodies for immuno-affinity chromatography." J. Immunol. Meth., 24, 305-309. Tijssen, P. (1985) "Practice and Theory of Enzyme Immunoassays." Vol. 15, Elsevier, New York. Nose, M. and Wigzell, H. (1983) "Biological significance of carbohydrate chains on monoclonal antibodies." Proc. Natl. Acad. Sci., 80, 6632-6636. Sox, H. C. and Hood, L. (1970) "Attachment of carbohydrate to the variable region of myeloma immunoglobulin light chains." Proc. Natl. Acad. Sci., 66(3), 975-982. Wallic, S. C., Katat, E. A. and Morrison, S. L. (1988) "Glycosylation of a VH residue of a monoclonal antibody against (1-6) dextran increases its affinity for antigen." J. Exp. Med., 168, 10-99-1109. Schneider, C., Newman, R. A., Sutherland, D. R. Asser, U. and Greaves, M. F. (1982) "A one-step purification of membrane proteins using a high efficiency immunomatrix." J. Biol. Chem., 257 (18), 10766-10769. Akerstrostrom, B., Brodin, T., Reis, K. and Bjorck. L. (1985) "Protein G: a powerful tool for binding and detection of monoclonal and polyclonal antibodies." J. Immunol., 135, 2589-2592. O'Shannesy, D. J. and Quarles, R. H. (1987) J. Immunol. Methods, 99, 153. Babashak, J. V. and Philips, T. M. (1988) J. Chromatogr., 444, 22. Sato, H., Kidake, T. and Hori, M. (1987) "Leakage of immobilized IgG from therapeutic immunoadsorbents." Appl. Biochem. Biotech., 15, 145-158. Sterns, D. J., Kurosawa, S., Sims P. J., Esmon, N. L. and Esmon, C. T. (1988) "The interaction of a Ca.sup.2+ -dependent monoclonal antibody with the Protein C activation peptide region," J. Biol. Chem., 63(2), 826-832. Orthner, C. L., Madurawe, R. D., Velander, W. H., Drohan, W. N., Battey, F. D., Strickland, D. K. (1989) "Conformational changes in an Epitope Localized to the NH.sub.2 -terminal Region of Protein C." J. Biol. Chem. 264(310 18781-18788. Sakuragawa, N., Shimizu, J., Kondo, K., Kondo, S., Niwa, M. (1986) "Studies on the effect of PEG-modified urokinase on coagulation-fibrinolysis using beagles." Thrombosis Research, 41, 627. Berger, Jr., H. and Pizzo, S. V. (1988) "Preparation of polyethylene glycol-tissue plasminogen activator adducts that retain functional activity: characteristics and behavior in three animal species." Blood, 71, 1641-1647. All references discussed or mentioned above or hereafter are incorporated by reference as if reproduced in full below.
Thus, there is a need for immunosorbents with improved binding efficiency, such that a high percentage of the antibodies bound or immobilized on a support are capable of maintaining avidity sufficient to attract and bind with proteins or haptens which they are capable of binding with when not bound to a support (or otherwise complexed). There is also a need for an inexpensive process for preparing immunosorbents having improved binding efficiency. An immunosorbent with improved binding efficiency is defined hereinafter as any immunosorbent in which greater than 30% of bound antibodies or bound ligands are active; active is defined hereinafter as the ability of the specific molecular recognition site on a ligand or the ability of the antitope on an antibody to be able to attract and bind to compounds or antigens.